The lung critically relies on the availability of lipoproteins to maintain adequate synthesis of surfactant by alveolar type II epithelial cells. Lipoproteins are a rich source of fatty acids, which serve as substrates for phosphatidylcholine (PC), the major lipid of surfactant. Lipoproteins also serve as potent post-translational activators of the rate-limiting enzyme required for surfactant PC synthesis, cytidylyltransferase (CT). Although much is known about post- translational control of CT, very little is known about transcriptional regulation of this enzyme. In addition, feedback control mechanisms that exist in the lung to maintain surfactant PC homeostasis under conditions of lipoprotein deficiency remain largely unknown. This proposal seeks to expand recent advances made in our laboratory showing that chronic lipoprotein deficiency increases CT activity and surfactant synthesis in primary type II cells by increasing CT mRNA synthesis. These results suggest that lipoprotein deprivation stimulates CT gene transcription as a novel compensatory or adaptive mechanism. These preliminary results led us to hypothesize that CT is regulated, in vitro and in vivo, at the transcriptional level. We propose to test this hypothesis by first assessing transcriptional regulatory activity using deletional and mutational analysis of the CT promoter to identify putative lipid (sterol)-sensitive enhancer elements that regulate this promoter in murine lung epithelial cell lines (MLE) in the setting of lipoprotein deprivation (AIM 1). We will next test our hypothesis in vivo by using recently generated CT promoter-reporter mice (AIM 2). These transgenic mice will be used to study constitutive and inducible CT transcription after lipoprotein deprivation that is achieved using nutritional, pharmacologic, and genetic approaches. Transgenic mice harboring specific CT promoter segments linked to the beta-galactosidase (beta gal) reporter gene will be used to determine the functional relevance of regulatory elements within the 5' flanking region of the CT promoter in vivo. Lungs will be analyzed for reporter expression by immunohistochemical staining, by immunoblotting, by assay of enzyme activity, and Northern analysis. Results will be correlated with endogenous CT expression and surfactant production. The unique contributions of this proposal impacting the field of surfactant metabolism include providing a springboard by which investigators can understand, for the first time, how the CT gene is regulated transcriptionally in vitro and in vivo. Ultimately, the results from these studies could lead to therapeutic strategies directed at modulating expression of this key surfactant enzyme at the molecular level.